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. 2018 Feb 16;293(7):2370-2380.
doi: 10.1074/jbc.RA117.000634. Epub 2017 Dec 18.

X-linked inhibitor of apoptosis protein (XIAP) is a client of heat shock protein 70 (Hsp70) and a biomarker of its inhibition

Laura C Cesa  1 , Hao Shao  2 , Sharan R Srinivasan  1 , Eric Tse  3   4 , Chetali Jain  5 , Erik R P Zuiderweg  3 , Daniel R Southworth  1   3   4 , Anna K Mapp  1   4   5 , Jason E Gestwicki  6
Affiliations

X-linked inhibitor of apoptosis protein (XIAP) is a client of heat shock protein 70 (Hsp70) and a biomarker of its inhibition

Laura C Cesa et al. J Biol Chem. .

Abstract

Heat shock protein 70 (Hsp70) and Hsp90 are molecular chaperones that play essential roles in tumor growth by stabilizing pro-survival client proteins. However, although the development of Hsp90 inhibitors has benefited from the identification of clients, such as Raf-1 proto-oncogene, Ser/Thr kinase (RAF1), that are particularly dependent on this chaperone, no equivalent clients for Hsp70 have been reported. Using chemical probes and MDA-MB-231 breast cancer cells, we found here that the inhibitors of apoptosis proteins, including c-IAP1 and X-linked inhibitor of apoptosis protein (XIAP), are obligate Hsp70 clients that are rapidly (within ∼3-12 h) lost after inhibition of Hsp70 but not of Hsp90. Mutagenesis and pulldown experiments revealed multiple Hsp70-binding sites on XIAP, suggesting that it is a direct, physical Hsp70 client. Interestingly, this interaction was unusually tight (∼260 nm) for an Hsp70-client interaction and involved non-canonical regions of the chaperone. Finally, we also found that Hsp70 inhibitor treatments caused loss of c-IAP1 and XIAP in multiple cancer cell lines and in tumor xenografts, but not in healthy cells. These results are expected to significantly accelerate Hsp70 drug discovery by providing XIAP as a pharmacodynamic biomarker. More broadly, our findings further suggest that Hsp70 and Hsp90 have partially non-overlapping sets of obligate protein clients in cancer cells.

Keywords: cancer biology; chaperone; chemical biology; heat shock protein 90 (Hsp90); protein–protein interaction.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
IAPs are selectively destabilized by Hsp70 inhibition. A, chemical structures of inhibitors. PES and JG-98 inhibit Hsp70, whereas AUY-922 and 17-DMAG inhibit Hsp90. B, Hsp70 and Hsp90 inhibitors reduce the growth of MDA-MB-231 cells, as measured by MTT assays. C, kinetics of Hsp70 and Hsp90 inhibitor-mediated anti-proliferative activity. Results are the average of experiments performed in triplicate. Error bars represent S.E. D, destabilization of IAPs occurs after treatment with Hsp70 inhibitors. MDA-MB-231 cells were treated for the indicated times. The blots shown are representative of at least two independent experiments. The error bars represent S.E.
Figure 2.
Figure 2.
Hsp70 binds XIAP. A, domains of human XIAP. B, XIAP is co-immunoprecipitated with Hsp70 in MCF7 lysates. Results are representative of experiments performed in triplicate. C, XIAP(120–356) binds Hsp70 in vitro, as measured by ELISA. Addition of JG-98 (10 μm) modestly weakens the interaction. Results are the average of three independent experiments performed in triplicate. Error bars are S.E. D, Smac mimetic SM164 blocks binding of Hsp70 to XIAP(120–356) by ELISA. Results are the average of three independent experiments performed in triplicate. Error bars represent S.E. E, purified Hsc70 and Hsp90a compete with Hsp72 for binding to XIAP in the ELISA format. Results are the average of triplicates, and error bars represent S.E. IP, immunoprecipitation; IB, immunoblot.
Figure 3.
Figure 3.
Hsp70 binds multiple sites in the BIR2 and BIR3 domains. A, location of predicted Hsp70-binding sites on BIR2 and BIR3 (Protein Data Bank codes 1C9Q and 1F9X) are shown in red. B, CD spectra for XIAP(120–356) wildtype and mutant proteins. Each reported spectrum is the average of six scans, subtracting the signal acquired for buffer alone. C, binding of XIAP(120–356) point mutants to Hsp70, as measured by ELISA. Results are the average of three independent experiments performed in triplicate. Error bars are S.E. D, XIAPY190E is partially resistant to degradation in response to JG-98. HeLa cells over-expressing the indicated XIAP point mutations were treated for 24 h. Blots are representative of two independent experiments, and quantification is from the average of biological replicates. Error bars are S.E. *, p < 0.06; ns, not significant. E, Hsp70 binds to XIAP and forms a stable, multimeric complex, as analyzed by SEC-MALS. Molecular weight standards were BSA and β-amylase. Results are representative of duplicates.
Figure 4.
Figure 4.
XIAP(120–356) binds Hsp70 through partially non-canonical interactions. A, canonical peptide, NRLLLTG, does not compete with XIAP(120–356) for binding to Hsc70, as measured by ELISA. B, Hsp70 binding to XIAP(120–356) is not classically nucleotide-dependent. The affinity is slightly tighter in the presence of ATP, rather than ADP. C, full-length Hsc70, Hsc70NBD, and Hsc70SBD compete with Hsp70 for binding to XIAP(120–356), as measured by ELISA. All results shown are averages of three independent experiments performed in triplicate, and error bars represent the S.E. Binding data were fit to the Langmuir binding isotherm, and competition data were fit to the Hill equation. D, 15N-1H TROSY-HSQC spectra of 35 μm Hsp70 SBD(395–507) without (black) or with (red) 200 μm NRLLLTG and 15N-1H TROSY-HSQC spectra of 100 μm Hsp70 SBD(395–507) without (blue) or with (red) 131 μm XIAP(120–356).
Figure 5.
Figure 5.
IAPs are potential biomarkers of Hsp70 inhibition in cancer cells. A and B, IAPs are de-stabilized by Hsp70 inhibition in HeLa (left) and MCF7 (right) cells (A), but not in IMR90 cells (B). Representative blots of at least two independent biological replicates are shown. Protein levels are quantified, and results shown are averages of two independent experiments. Error bars represent S.E. C, treatment with JG-98 results in loss of IAP protein levels in MCF-7 mouse xenografts.
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